THE AQUEOUS PORE THROUGH THE TRANSLOCON HAS A DIAMETER OF 40-60 ANGSTROM DURING COTRANSLATIONAL PROTEIN TRANSLOCATION AT THE ER MEMBRANE

Eukaryotic secretory proteins are cotranslationally translocated throu gh the endoplasmic reticulum (ER) membrane via aqueous pores that span the lipid bilayer. Fluorescent probes were incorporated into nascent secretory proteins using modified Lys-tRNAs, and the resulting nascent chains were sealed off from the cytosol in fully assembled translocat ion intermediates. Fluorescence quenching agents of different sizes we re then introduced into the ER lumen in order to determine which were small enough to enter the pore and to quench the fluorescence of probe s inside the ribosome and/or the pore. These accessibility studies sho wed that the aqueous pore in a functioning translocon is 40-60 Angstro m in diameter, making it the largest hole observed to date in a membra ne that must maintain a permeability barrier.